Although progress in transplantation has been achieved during the past decade, many patients waiting for organ transplant face death due to a severe shortage of donor organs. This shortage of organs has engendered a heightened interest in the field of xenotransplantation. Organs from swine are viewed as the most promising candidates for transplants into humans. The major barrier for successful xenotransplantation between discordant species is hyperacute rejection. However, progress in the understanding of hypemcute rejection and approaches to abrogate it have made it likely that in the very near future, hyperacute rejection will be overcome. It will then be necessary to define other mechanisms of xenograft rejection mediated by cellular and humoral pathways of the immune system, including the role of porcine endogenous viruses (PERV) in eliciting human immune responses to porcine organs. To accomplish these goals we will: 1) Characterize the unique cross reaction identified between HLA and SLA antigens using HLA allo-antibodies from patients awaiting transplantation. We will purify anti-HLA from natural xenoreactive antibodies and define their reactivity to porcine cells using both complement-dependent cytotoxicity and immunofluorescence assays. Another approach will use SLA allele-transfected human cells as targets. 2) characterize the cellular and molecular changes in porcine aortic endothelial cells (PAEC) brought about by the ligation of SLA molecules by anti-HLA antibodies. Using purified anti-HLA antibodies we will ligate SLA molecules on PAEC, and signaling cascades will be analysed using biochemical and molecular approaches. We will also determine the molecules involved in the signaling by immunoprecipitation. 3) Determine the consequence of PERV infection for the human immune response to porcine xenografts. We will define the PERV derived peptide motifs which are recognized by human CD8+ T cells in the context of SLA molecules. These studies will be done by pulsing SLA transfected human cells with synthetic nanomer peptides derived from PERV. We will also determine whether human CD8+ T cells can be generated against PERV infected human cells. We will also determine whether PERV infection of human cells results in stable integration of the viral genome in host chromosome by RELAP and real time PCR of PERV infected cells, whether PERV integrase can catalyze the integration by in vitro end nuclease and integration assays and study the effect of this integration on activation of human T lymphocytes by analyzing activation markers by flow cytometry and analyzing the cytokine secretion pattern by ELISPOT. These studies will not only define the role of PERV in eliciting immune responses against xenografts but also towards autoimmunity. Our long term goals are to develop new approaches to prevent or to treat human anti-porcine xenograft rejection based on our knowledge gained in this project in understanding the immunological mechanisms of human anti-porcine cellular responses.